The long-term goal of this project is to study the nature of differences in the post-transcriptional modification of transfer RNAs which occur in certain cells, determine what regulates the modification, and understand the role of the modified tRNA. Bacillus subtilis 168 is employed as a model system in which tRNA changes can be studied within the context of a differentiation organism. We have previously shown that post-transcriptional modifications of tRNAs represent a significant phenomenon during development in B. subtilis. The present application concentrates on determination of primary sequences of unique tRNAs by the "rapid-print out" sequence technique and identification of modified nucleosides by standard biochemical procedures and also by immunochemical methods. In addition to these structural studies, isoaccepting species, which differ only in a post-transcriptional modification, will be tested for codon specificity in an in vitro protein synthesizing system. We further propose to purify and characterize the enzyme system in B. subtilis which is responsible for 2-thiomethylation of N6-(delta 2-isopentenyl)adenosine, since regulation of that enzyme activity is the cause of the difference in modification of at least one set of isoaccepting tRNAs.